The aim of the proposed research is to further our understanding of the contractile proteins in platelets and to elucidate their role in platelet function. In preliminary studies a 90,000 dalton polypeptide, produced by calcium-dependent proteolysis during platelet activation, has been identified. This polypeptide is associated with platelet cytoskeletons. Since this polypeptide is only produced under conditions in which the transmembrane glycoproteins IIb and III are also associated with cytoskeletons, the hypothesis has been formulated that it is involved in attaching actin filaments to the inner aspect of plasma membranes. Such a function would allow force, generated by actin filaments within platelets, to be transmitted to fibrin, bound extracellulary to glycoproteins IIb and III, thus allowing clot retraction to occur. Experiments are proposed to characterize the biochemical properties of this new 90,000 dalton polypeptide, to identify its binding site on cytoskeletons, membranes or other subcellular organelles and to establish its role in platelet function. The protein will be purified and used to generate an antibody for the identification of its precursor, for its quantitation in subcellular fractions and for its immunocytochemical localization. Binding of purified 90,000 dalton polypeptide to both cytoskeletons and membranes will be measured using purified radiolabeled protein. If specific binding to these subcellular fractions is detected, binding to potential receptors such as purified actin filaments and glycoproteins IIb and III will be measured. The role of the 90,000 dalton polypeptide in platelet responses will be determined by correlation of its production during stimulation of platelets by a variety of agents to the platelets responses to these agents. The effects of inhibitors of its production will be correlated with the effects of these inhibitors on platelet responses. By increasing our knowledge of the biochemical changes that occur during platelet stimulation, these experiments will further our knowledge of the role of platelets in hemostasis and identify potential ways by which abnormal platelet function can be controlled.